Tumor-associated macrophages (TAM) have recently attracted much attention as they play key roles in tumor spread and in response to therapy: TAM substantially accelerate the progression of untreated tumors; they also markedly influence the efficacy of anticancer drugs, including checkpoint blockade immunotherapies. Furthermore, targeting TAM themselves, e.g. through the colony-stimulating factor 1 receptor (CSF-1R), can effectively control the progression of advanced tumors. However, TAM show considerable plasticity by assuming opposing phenotypes and functions that can be either tumoricidal (e.g. M1-like cells) or tumorigenic (e.g. M2-like cells) and currently there is a significant knowledge gap on how TAM function in vivo and how these cells' activities can be harnessed to improve anticancer therapy. This is largely because most of our knowledge on TAM has come from histological and in vitro profiling studies, which are are invaluable methodological approaches but cannot address how TAM functions are integrated within the dynamic tumor microenvironment (TME). The goal of this project is to perform high resolution imaging??in combination with broader coverage imaging and complementary readouts??to reveal fundamental aspects of TAM and other myeloid subtypes during tumor progression in vivo. Further, we aim to use imaging to uncover how most recent treatments (e.g. anti-PD-(L)1 immune checkpoint blockers) and new promising approaches (CSF-1R inhibitors) affect TAM functions in vivo and in turn how drug effects on TAM impact treatment efficacy. Specifically, to advance our knowledge of TAM subtypes and other myeloid cells, we will combine: i) new mouse models and reporters for these cells; ii) single cell resolution intravital imaging of tumor environments; iii) broad coverage microscopic imaging and other ex vivo readouts; and iv) new computational analysis. Taken together, findings from this research proposal should not only deepen our understanding of mechanisms regulating myeloid cell responses but also help uncover new opportunities for next-generation immunotherapeutic strategies and TAM imaging. This immunotherapy imaging project brings together tumor immunology (Pittet) and imaging (Weissleder) expertise from two different laboratories. We are well positioned to achieve our goals, having demonstrated the feasibility of the experiments outlined in this application.